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1590/S2179-975X3615
SoCarlos UFSCar, Rodovia Joo Leme dos Santos, Bairro do Itinga, CEP 18052-780, Sorocaba, SP, Brazil
e-mail andrecas@ufscar.br; iolanda@ufscar.br
3
Departamento de Microbiologia Agrcola, Escola Superior de Agricultura Luiz de Queiroz ESALQ,
Universidade de So Paulo USP, Av. Pdua Dias, 11, CEP 13418-900, Piracicaba, SP, Brazil
e-mail: emilianaromagnoli@usp.br
4
Departamento de Hidrulica e Saneamento, Escola de Engenharia de So Carlos, Universidade de
SoPaulo USP, Av. Tabalhador So Carlense, 400, CEP 13566-590, So Carlos, SP, Brazil
e-mail: Calijuri@sc.usp.br
Figure 1. Location of Itupararanga Reservoir and sampling points. P1 = dam and P2 = Entrance.
Table 1. Geographic coordinates of the sample points. GCT GG) (Muyzer et al., 1993) were used and
Sample Points Geographic coordinates quantified with Platinum Quantitative PCR
Entrance 23 37 3.8 S and 47 13 41.4 W super mix UDG (Invitrogen). Each reaction was
Dam 23 36 44.6 S and 47 23 40.9 W performed in a volume of 25L containing 1L
sample DNA, 0.25 L BSA and 1 L of each primer.
2.2. Microbial analysis The PCR cycling conditions were as follows: 3min
at 95C, 35 cycles of 30 sec at 94C, 30 sec at 55C,
First, the water samples (1L) were passed
30 sec at 72C and a final extension at 72C for 3
through filter membranes of 0.45 m pore size
minutes.
(Milipore model HAQG047S3) and then through
For archaeal 16S rDNA, the primers 340F (CCC
membranes of 0.22 m pore size (both of 0.47 mm
TAY GGG GTG CAS CAG) and 1000R (GAG
diameter, Milipore model GSWP047S0). The DNA
of the water was extracted from the membranes ARG WRG TGC ATG GCC) (Gantner et al.,
using a metagenomic DNA isolation kit for water 2011) were used and quantified with Platinum
(Epicentre laboratories), and from the sediments Quantitative PCR super mix UDG (Invitrogen).
with the Ultraclean soil DNA isolation kit (Mobio Each reaction was performed in a volume of
laboratories), according to the manufacturers 25L containing 1L sample DNA, 0.25 L
instructions. The DNA concentrations were BSA, 50pmol MgCl and 10 pmol of each primer.
determined by measuring absorbance at 260 nm ThePCR cycling conditions were as follows: 2min
(A260). For pure DNA, the ratio of A 260 to at 98C, 35 cycles of 30 sec at 95C, 30 sec at
A 280 is between 1.8 and 1.9 (Wilhelm et al., 57C, 90 sec at 72C and a final extension at 72C
2003). The intact DNA was then confirmed by for 7minutes.
electrophoresis on a 1% agarose gel. The copy number of 16 S rDNA in the samples
was determined in duplicate and all PCR assays
2.3. Quantitative polymerase chain reaction analysis included a negative control containing no DNA to
Quantitative real time PCR (qPCR) assays were test for potential contamination. The abundance
used to measure the abundance of Bacteria and of target genes in each sample was estimated by
Archaea genes in the water column. To measure parallel quantitative PCR assay of the dilutions of
bacterial 16S rDNA, the primers P1 (CCT AGC the standards and by comparing threshold cycles
GGA GGC AGC AG) and P2 (ATT ACC GCG (Ct), which is the cycle at which the fluorescence
414 Soares, L.A.etal. Acta Limnologica Brasiliensia
intensity surpasses the level at which amplification The center of the Itupararanga Reservoir is
enters a logarithmic growth phase, obtained in each characterized by a stratification period in the
PCR run with DNA standards. summer and a homogeneous vertical profile during
the winter. Contrastingly, the peripheral regions of
2.4. Polymerase chain reaction/electrophoresis in gel
the reservoir were stratified during both periods
of denaturant gradient
(Cunha & Calijuri 2011a; Cunha et al., 2012;
The bacterial and archaeal communities from Miwaetal., 2011).
the water column were evaluated by Polymerase The nutrient concentration also varied along
Chain Reaction and Denaturing Gradient Gel the water column. Organic and inorganic solids
Electrophoresis (PCR-DGGE). The 16S rRNA increased with depth at the entrance and decreased
genes from the Archaea and Bacteria domains were with depth at the dam. This profile is in agreement
amplified using primers: 1100F (with GC clamp) with the profile proposed by Thornton (1990),
and 1400R (Kudo et al., 1997); 968 FGC who divided a tropical reference reservoir into three
and 1392R (Nielsen et al., 1999), respectively. zones. The first zone (riverine zone) occurs at the
Denaturing gradient gel electrophoresis (DGGE) entrance of effluent and is characterized by greater
was performed using the DcodeTMUniversal water flow and a greater amount of allochthonous
Mutation Detection System (Bio-Rad Laboratories, organic matter. The next zone (transitional zone) is
Hercules, CA, USA) in accordance with the characterized by intermediate conditions, and the
manufacturers instructions. The PCR products were last zone (lacustrine zone) is characterized by lower
electrophoresed in TAE buffer (1x) at 75V for 16h at water flow and autochthonous organic matter.
60C on a polyacrylamide gel (7.5%) containing a Nitrogen, nitrate, nitrite, orthophosphate and
linear gradient of the denaturant from 45% to 65%. phosphorus concentrations were higher in the
The polyacrylamide gel was stained with ethidium entrance zone than at the dam (Table2). According
bromide for 20 min and then visualized using an to Beghellietal. (2014) these nutrients are received
Eagle Eye TM III densitometer (Stratagene) with from tributary rivers.
254 nm UV exposures, coupled to a personal The abundance of archaea and bacteria in the
computer using Eagle Sight software. sediment was greater than in the water column
2.5. Statistical analysis (Figure 2), which may be associated with higher
organic matter, nutrient concentrations and anoxic
Canonical Correspondence Analysis (CCA) was conditions of sediments.
used to group the chemical and microbial properties. The abundance of bacteria was greater in the
Correlation analysis of microbial abundances sediment from entrance (2.56 x 10 9cell. g-1 of
of samples from each site (dam and entrance) sediment) than from the dam (6.09 x 108cells. g-1
was performed. Pearsons similarity coefficient of sediment) probably due to the high nutrients
was calculated using BioNumerics software, available. Beghellietal. (2012), however, classified
version2.5. The Shannon-Wiener diversity index the entrance point as eutrophic and the remainder
was calculated based on the intensity of the DGGE of the reservoir as mesotrophic. Tertova et al.
bands and on the height of the densitometric curve (2013) observed a similar profile in the eutrophic
peaks, according to Abreuetal. (2010). Lake Vartsjarv situated in southern Estonia,
finding by flow cytometry a great abundance of
3. Results and Discussion bacteria in the river (1.6 x 109.g-1 dry weight) and
The water column was chemically and thermally middle sediments of the lake (1.8 x 109cells. g-1
stratified at both sampling sites. Surface water dry weight) and low abundance in the littoral zone
temperature at the dam and entrance was 24.4 C (8.0 x 107.g-1 dry weight).
and 23.1, respectively, decreasing to 22.7C and Maintingueretal. (2011) found hydrogenotrophic
22.3C at the bottom of the reservoir. Dissolved bacteria such as bacillus and endospore producers
oxygen concentration decreased at the bottom of the of hydrogen in the Itupararanga sediment and
reservoir, and while no anoxic zone was observed, quantified the acidogenic bacteria to a more
hypoxia was identified (1.4 mg.L-1 and 2.5 mg.L-1, probable number (4.3x102 NMP.mL-1 equivalent
dam and entrance, respectively). The lower oxygen to 3.3x105 gSTV-1 cells).
concentrations of the deeper layers are associated This longitudinal pattern, with a higher trophic
with the stratification of conditions (Salcheretal., status in the riverine zone of reservoirs where the
2011). inflow rivers are found, is expected due to the
2015, 27(4), 411-420 Distribution of Archaeal and Bacterial communities... 415
Table 2. Mean of environmental variables in entrance zone and dam of Itupararanga Reservoir.
Dam Entrance
Variables
0.0 m 2.0 m 4.0 m 10.0 m 15.5 m 0.0 m 4.0 m 8.0 m
Temperature (C) 24.39 24.26 24.16 23.34 22.7 23.14 22.62 22.28
pH 7.34 7.18 7.09 6.15 6.45 6.92 6.42 6.33
Dissolved oxygen (mg.L-1) 8.29 8.65 8.3 5.6 1.4 5.65 3.6 2.5
Conductivity (S.cm-1) 67 66 69 55 98 96 100 101
Total susp. solids (mg.L-1) 5.7 4.65 5.25 3.1 1.75 5.9 6.8 8.65
Organic susp. solids (mg.L-1) 1.7 1 2.25 1.25 0.45 2.8 3.85 5.5
Inorganic susp. solids (mg.L-1) 4 3.65 3 1.85 1.3 3.1 2.95 3.15
Alkalinity (mgCaCO3.L-1) 0.94 1.17 1.23 2.67 2.11 2.4 2.18 2.22
Orthophosphate (g.L-1) 0.0022 0.0028 0.0025 0.0024 0.0026 0.006 0.0071 0.0078
Nitrate (mg.L-1) 0.35 0.39 0.4 0.39 0.39 0.61 0.57 0.61
Nitrite (mg.L-1) 0.66 0.52 0.565 0.58 0.64 4.63 4.05 6.55
Ammonia (mg.L1) 5.4 5.2 4.2 25.6 51.04 4.37 3.67 10.9
Dissolved Phosphorus (mg.L-1) 0.37 0.53 0.43 0.51 0.57 1.06 1.27 1.73
Nitrogen Kjeldahl (mg.L-1) 0.599 0.571 0.586 0.265 0.223 0.432 0.432 0.655
Chlorophyll (g.L-1) 0.28 0.32 0.417 0.261 0.272 0.346 0.342 0.267
Figure 2. Relative abundance of Archaea and Bacteria community into the water column from (a) Dam, (b) Entrance
and (c) Sediment of Itupararanga Reservoir.
reduction of flow and increased sedimentation probably due to the fact that the metabolic plasticity
(Nogueiraetal., 1999; Thornton 1990). The higher of the bacterial communities is generalist, while
density of Bacteria and Archaea in the sediment archaeal communities are specialist and more
of this region can be an expected pattern in other adapted to stress conditions (Smetietal., 2013).
reservoirs. The DGGE profiles for the archaeal and
Bacteria were more abundant than archaea bacterial communities from the entrance and dam
in both the water and sediment samples, most are shown in Figure3. The archaeal communities
416 Soares, L.A.etal. Acta Limnologica Brasiliensia
Figure 3. DGGE profile for Archaea (a) and Bacteria (b) Domain. Entrance E1= 0m; E2 = 4.0 m and E3 = 8.0 m;
Dam D1 = 0m, D2 = 2.0m, D3 = 4.0m, D4= 10.0m and D5 15.5 m.
were separated into two groups according to the The similarity between the archaeal communities
sampled region (entrance and dam). On the other of the entrance and the dam was 78%. In this
hand, lower variability was observed for the bacterial sense, the archaeal community was distributed
than the archaeal communities. homogenously in the Itupararanga Reservoir.
Major similarities were observed for the archaeal On the other hand, the bacterial similarities
communities along the water column and along between the surface and bottom were 42% and 52%
the reservoir. The similarity between the archaeal at the dam and entrance, respectively, indicating
communities from the surface and the bottom was that the chemical and thermal stratification of the
60% and 94% at the entrance and at the dam, water column had an important influence on the
respectively, indicating that the environmental bacterial community.
changes had more influence on the distribution The microbial diversity established from the
of archaeal communities at the entrance than at DGGE profiles was measured with the Shannon
the dam. diversity index (H). At the entrance point (P1) the
The greater similarity between the archaeal diversity increased from the surface to the bottom
communities from the surface and the bottom at (Table3).
the dam (94%) than at the entrance (60%) was The archaeal diversity increased from the surface
probably due to the greater stability in the dam to the bottom at the entrance (1.47 to 2.50) and
region than at the entrance, despite the thermic dam points (1.61 to 3.95). The same profile
and oxygenic stratification. was observed for bacterial diversity at the dam
2015, 27(4), 411-420 Distribution of Archaeal and Bacterial communities... 417
Figure 4. Correspondence Canonical Analysis (CCA) between environmental- Chlorophyll (Chl), Organics suspended
solids (OSS), dissolved oxygen (DO), Profundity (Prof ) and ammonia (NH4) and biological variables- Archaeal
density (ADe) Bacterial density (BDe), Archaeal diversity (ADi) and Bacterial diversity (BDi) from Itupararanga
Reservoir, SP, Brazil.
418 Soares, L.A.etal. Acta Limnologica Brasiliensia
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